Site-specific in vitro mutagenesis was used to direct serine for cysteine substitutions within the sequence of human interferon-alpha 1 (IFN-alpha 1). Antiviral specific activities and antiproliferative activities of IFN-alpha 1 analogs, expressed in M13 as fusion proteins, were assessed following purification by monoclonal antibody affinity chromatography. Based on analysis of IFN-alpha 2, IFN-alpha 1 contains two disulfide bridges between cysteine residues 29 and 139 and cysteine residues 1 and 99. IFN-alpha 1 also contains a fifth cysteine residue at position 86. The series of serine for cysteine substitutions performed indicated that IFN-alpha 1 molecules unable to form the residue 29 to residue 139 disulfide bridge have substantially reduced antiviral and antiproliferative activities, IFN-alpha 1 molecules unable to form the residue 1 to residue 99 disulfide bridge have only marginally altered antiviral and antiproliferative activities, the low antiviral activity of IFN-alpha 1 compared with other human IFN-alpha subtypes is not due to the formation of nonnative disulfide bridges involving the fifth cysteine residue at position 86, which the other subtypes lack, and (iv) the reduced biological activities of certain analogs may be due to the formation of nonnative disulfide bridges.